1. Field of the Invention
The present invention generally relates to a method of using a recently discovered class of polypeptides for boosting red blood cell (erythrocyte) production and, more particularly, to using an atrial natriuretic factor (ANF) at a low concentration to potentiate the activity of erythropoietin (Epo).
2. Description of the Prior Art
In the United States alone, several million people suffer from anemia secondary to renal failure, chronic inflammatory disease and malignancies. Many hypotheses have been proposed for explaining the mechanism behind the development of anemia, and they include the blockade of iron in the reticuloendothelial system due to abnormal iron metabolism, impaired iron release, spleen involvement, impaired Epo production, hemolysis, and disorders in hemopoietic stem cells. Treatment of chronic anemia with blood transfusions may become unacceptable because of the risk of viral infections, such as AIDS or hepatitis, and other side effects, such as transfusion reaction or iron accumulation.
Epo is a large glycoprotein hormone synthesized in the kidney which affects the terminal stages of erythroid differentiation by enhanced cell division and maturation (see. Iscove, Cell Tissue Kinet. 10: 323, (1977)). Epo stimulates the proliferation of the late erythroid progenitor known as colony forming unit-erythroid (CFU-E) which are detectable by in vitro cell culture but not by microscopy, and morphologically recognizable erythroid cells which are derived from CFU-E after cell division and maturation (see, Jelkinan, Rev. Physiol. Biochem. Pharmacol 104: 139, (1986)).
Humoral control of the early stages of erythropoiesis is not as well understood. In vitro stimulation of the early erythroid progenitor known as burst forming unit-erythroid (BFU-E) has been observed in the presence of three species of molecules. Interleukin-3 (IL-3), also called multipotential growth factor (Multi-CSF), which acts on many hemopoietic precursors, stimulates BFU-E formation in vitro (see, Sieff, J. Clin. Invest. 79: 1549, (1987)). Granulocyte-macrophage colony stimulating factor (GM-CSF), which is thought to act mainly on granulocyte and macrophage precursors, stimulates BFU-E in vitro if cultures are initiated in the presence of this factor and Epo is added three to five days later (see, Donahue et al., Blood 66: 1479, (1985)). Erythroid potentiating activity factor (EPA) stimulates the growth of both BFU-E and CFU-E in vitro from both human and murine marrow and stimulates colony formation by the K-562 human erythroleukemic leukemia line cells (see, Gasson et al., Prog. Clin. Biol. Res. 184: 95, (1985), Gasson et al., Nature 315: 768, (1985), and Fraser et al., Blood 71: 104, (1988)). In vivo enhancement of erythropoiesis has been observed after administration of EPA to experimental animals (see, Niskanen et al., Clin Res 35: 429A, (1987) and Niskanen et al., Blood, (in press), (1988)). IL-3, GM-CSF and EPA are all large proteins that are difficult to produce in large quantities and are not easily introduced into the bloodstream by noninvasive means.
The sequence of red blood cell production can be summarized generally in the following manner. BFU-E originate from multipotential progenitor cells and give rise to CFU-E. The CFU-E then give rise to nucleated erythroid cells. The nucleated erythroid cells give rise to reticulocytes, and the reticulocytes give rise to red blood cells.
U.S. Pat. No. 4,732,889 to Cynshi et al discloses using Epo to treat anemia found in patients suffering from rheumatoid arthritis. Studies performed on adjuvant-induced arthritis rats showed that rats which had been injected intraperitoneally with human urinary derived Epo or cell derived recombinant Epo had significantly improved erythrocyte counts as compared with a control group. Currently, the treatment of choice for patients suffering from anemia is replacement therapy with recombinant Epo (see, for example, Eschbach et al., New Eng. J. Med. 316: 73-78, (1987) and Erslev, New Eng. J. Med. 316: 101-103, (1987)). Some of the disadvantages of replacement therapy with recombinant Epo include the high cost of Epo, the necessity of applying Epo by injection, and the possibility of developing antibodies which destroy Epo. Development of the antibodies may occur because large proteins, like Epo, are likely to be immunogenic.
Hypoxia is a deficiency in the amount of oxygen reaching bodily tissues. Stimulation of red blood cell production following hypoxia has been attributed to enhanced synthesis of Epo and its subsequent release from the kidney into the bloodstream (see, Jelkinan, Rev. Physiol. Biochem. Pharmacol. 104: 139, (1986)). ANF is another hormone released in response to low oxygen pressure (see, Baertschi et al., Biochem. Biophys. Res. Comm. 140: 427, (1986), and Baertschi et al., Am. J. Physiol., (in press), (1988)). ANF peptides are active hormonal substances which are synthesized in cardiac atria. Peptides which have been obtained from heart muscle have also been referred to as cardionatrin (de Bold et al., Life Sci., 33 297, (1983)), atriopeptin (Currie et al., Science 223 67, (1984)), and auriculin (Atlas et al, Nature 309: 717, (1984)). ANF is a family of polypeptides, all of which have a common amino acid sequence, but differ in length by the presence or absence of 1-8 amino acids on the amino or carboxyl termini (N-terminal or C-terminal, respectively).
U.S. Pat. No. 4,663,437 to de Bold discloses that extracts of ANF peptides play a role in extracellular fluid volume regulation. Shortly after administration, the extract enhances urinary flow and increases urinary sodium, potassium, and chloride excretion. U.S. Pat. No. 4,607,023 to Thibault et al discloses synthesized ANF peptides having diuretic, natriuretic, vasorelaxant, hypotensive or anti-hypertensive properties. These synthesized ANF peptides are administered systemically, either by intravenous, subcutaneous, or intramuscular injection, or by sublingual or nasal administration. U.S. Pat. No. 4,652,549 to Blaine discloses that ANF peptides can act as cardiac anti-hypertrophic agents. In addition, ANF peptides allow intravascular fluid to exit across capillaries leading to acute hematocrit rise (see, Fluckiger et al., Am. J. Physiol. H251:H880, (1986)).